Acetaminophen (APAP) hepatotoxicity is currently the most common cause of acute liver failure in the United States. Our long-term goal is to elucidate the function of liver innate immune system in the development of murine APAP hepatotoxicity. The specific hypothesis is that in addition to upstream factors involved in and responding to APAP metabolism, the downstream factors that lead to modulation of liver innate immunity may affect the individual's risks for developing severe APAP hepatotoxicity. Natural killer (NK) and NKT (NK cells with T cell receptors) cells, which are key components of the innate immune system, are highly enriched in the liver in both mouse and human. We have found that NK/NKT cells play a pivotal role in determining the progression and severity of APAP hepatotoxicity based on the observations that 1) depletion of these cells in vivo significantly protected mice from APAP-induced liver injury and improved mouse survival, 2) the protection was correlated with inhibition of inflammatory cytokine/chemokine production and hepatic neutrophil infiltration, 3) as the downstream event of NK/NKT activation, recruited hepatic neutrophils also contribute to liver injury. Based on these observations, the overall goal of this proposal is to further elucidate the activation modes of NK/NKT cells and its interplay with Kupffer cells, and the molecular and cellular mechanisms of cytotoxicity against mouse hepatocytes as they pertain to APAP hepatotoxicity in mice. The specific aims are to: 1) Determine up-stream activator(s) of NK/NKT cell activation. The role of cytokines and/or receptor recognition will be examined. 2) Further define the importance of NK/NKT cells in the interplay of liver innate immune system in response to APAP toxicity. The critical role of NK/NKT cell-derived IFN? will be validated by adoptive transfer of hepatic NK/NKT cells from wild type mice into IFN?-deficient mice. 3) Determine if NK/NKT cells are cytotoxic to hepatocytes and the molecular mechanism. Whether APAP sensitizes hepatocytes to cytotoxicity of innate immune cells will be investigated. It is hoped that mechanistic insights from these studies will be relevant to understanding the basis for susceptibility to idiosyncratic drug-induced liver disease. Public Health Relevance: Acetaminophen (APAP) is an over the counter medication used by many people. The liver toxicity due to APAP is currently the most common cause of acute liver failure in the United States. It is thus of great interest and importance to understand the mechanism of action leading to liver toxicity so that better therapeutics can be developed to prevent or ameliorate drug-induced liver injury.